While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33−/− mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33−/− mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33−/− mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals.While anti-miR-33 therapies offer promise for treating cardiovascular disease, deletion of miR-33 causes obesity and metabolic dysfunction. Price et al. elucidate how miR-33 deficiency affects metabolic functions in different tissues. Rather than finding that dysregulation of SREBP-1-mediated lipid synthesis primarily drives these effects, they demonstrate that these changes depend on increased food consumption.Consistent with previous work (Horie et al., 2013), we find that feeding of miR-33−/− mice with a HFD results in a dramatic increase in BW compared to control animals, especially during the first few weeks of HFD feeding. CD animals lacking miR-33 also developed increased BW although at a much more gradual rate (Figures 1A and 1B). This increase in BW was primarily due to increased fat mass, which was significantly elevated in 7-month-old mice on either a CD or HFD (Figure 1C). We further show that HFD fed miR-33−/− mice have impaired regulation of glucose homeostasis as demonstrated by an impaired ability to restore blood glucose levels during a glucose tolerance test (GTT) (Figure S1A). While CD-fed animals did not have any differences in the ability to regulate blood glucose levels during a GTT (Figure S1B), these mice have nearly a 2-fold increase in fasted insulin levels suggesting that increased insulin production may be compensating for impaired insulin sensitivity during this test (Figure S1C). To explore this in more detail, we performed hyperinsulinemic-euglycemic clamp studies to directly assess responsiveness to insulin in different tissues. Consistent with the elevated fasted plasma insulin levels, we find that the glucose infusion rate needed to maintain normal blood glucose concentrations after insulin administration was dramatically reduced in miR-33−/− mice (Figures 1D and S1D), indicating an overall impairment in insulin stimulated glucose uptake (Figure 1E). We further demonstrate that the capacity of both skeletal muscle and WAT to take up glucose in response to insulin is impaired, as incorporation of 2-deoxyglucose, a non-metabolizable form of glucose, was significantly reduced in miR-33−/− mice (Figures 1F and 1G). Similarly, the ability to suppress insulin-stimulated glucose production in the liver and hydrolysis of FFA from WAT was also dramatically impaired in miR-33−/− mice (Figures 1H and 1I). Together, these findings indicate that even on a CD, mice lacking miR-33−/− develop insulin resistance in numerous key metabolic tissues.For microarray analysis, the Laboratory of Genetics and Genomics, NIA IRP, and NIH performed the sample QC, labeling, hybridization, feature extraction, and GEO submission (Dr. Elin Lehrmann) and also provided bioinformatic analysis (Dr. Yongqing Zhang) within the microarray facility directed by Dr. Kevin Becker. This work was supported by grants from the NIH (R35HL135820 to C.F.-H.; R01HL105945 and R01HL135012 to Y.S.; 5F32DK10348902 to N.L.P.; and R01DK40936 to G.I.S.), the American Heart Association (16EIA27550005 to C.F.-H.; 16GRNT26420047 to Y.S.; and 17SDG33110002 to N.R.), the American Diabetes Association (1-16-PMF-002 to A.C.-D.), and the Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research MIRVAD (to C.F.-H.).DATA AND SOFTWARE AVAILABILITYThe accession number for the microarray data reported in this study is GEO: GSE109055.SUPPLEMENTAL INFORMATIONSupplemental Information includes seven figures and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.01.074.DECLARATION OF INTERESTSThe authors declare no competing interests.